Abstract
Epithelial organoid monoculture is a powerful tool to model stem cell dynamics in vitro. However, extensive efforts have recently revealed various niche players and their significant roles in regulating epithelial stem cells. Among these niche components, fibroblasts have been heavily recognized in the field as a critical niche signal secretor. Thus, understanding the roles of fibroblasts in epithelial dynamics has become increasingly relevant and crucial. This propels the development of approaches to coculture epithelial 3D organoids with fibroblasts to model epithelial-fibroblast crosstalk in vitro. Here, we describe a stepwise coculture method to isolate and culture primary intestinal fibroblasts and epithelial organoids together. Aligned with the recent literature, our coculture protocol allows for primary intestinal fibroblast support of epithelial organoid growth.
References
Sumigray KD, Terwilliger M, Lechler T (2018) Morphogenesis and compartmentalization of the intestinal crypt. Dev Cell 45(2):183–197 e185. https://doi.org/10.1016/j.devcel.2018.03.024
Barker N et al (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449(7165):1003–1007. https://doi.org/10.1038/nature06196
Sangiorgi E, Capecchi MR (2008) Bmi1 is expressed in vivo in intestinal stem cells. Nat Genet 40(7):915–920. https://doi.org/10.1038/ng.165
Yan KS et al (2012) The intestinal stem cell markers Bmi1 and Lgr5 identify two functionally distinct populations. Proc Natl Acad Sci USA 109(2):466–471. https://doi.org/10.1073/pnas.1118857109
Yan KS et al (2017) Intestinal Enteroendocrine lineage cells possess homeostatic and injury-inducible stem cell activity. Cell Stem Cell 21(1):78–90 e76. https://doi.org/10.1016/j.stem.2017.06.014
Sato T et al (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459(7244):262–265. https://doi.org/10.1038/nature07935
Ootani A et al (2009) Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat Med 15(6):701–706. https://doi.org/10.1038/nm.1951
Miyoshi H, Stappenbeck TS (2013) In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture. Nat Protoc 8(12):2471–2482. https://doi.org/10.1038/nprot.2013.153
Levin G et al (2020) Production, purification and characterization of recombinant human R-spondin1 (RSPO1) protein stably expressed in human HEK293 cells. BMC Biotechnol 20(1):5. https://doi.org/10.1186/s12896-020-0600-0
Lei NY et al (2014) Intestinal subepithelial myofibroblasts support the growth of intestinal epithelial stem cells. PLoS One 9(1):e84651. https://doi.org/10.1371/journal.pone.0084651
McCarthy N et al (2020) Distinct mesenchymal cell populations generate the essential intestinal BMP signaling gradient. Cell Stem Cell 26(3):391–402 e395. https://doi.org/10.1016/j.stem.2020.01.008
Kraiczy J et al (2023) Graded BMP signaling within intestinal crypt architecture directs self-organization of the Wnt-secreting stem cell niche. Cell Stem Cell 30(4):433–449 e438. https://doi.org/10.1016/j.stem.2023.03.004
McCarthy N et al (2023) Smooth muscle contributes to the development and function of a layered intestinal stem cell niche. Dev Cell 58(7):550–564 e556. https://doi.org/10.1016/j.devcel.2023.02.012
Staab JF et al (2020) Co-culture system of human enteroids/colonoids with innate immune cells. Curr Protoc Immunol 131(1):e113. https://doi.org/10.1002/cpim.113
Maruyama M et al (2023) Establishment of a novel in vitro co-culture system of enteric neurons and Caco-2 cells for evaluating the effect of enteric nervous system on transepithelial transport of drugs. Int J Pharm 633:122617. https://doi.org/10.1016/j.ijpharm.2023.122617
Psichas A et al (2017) Mixed primary cultures of murine small intestine intended for the study of gut hormone secretion and live cell imaging of enteroendocrine cells. J Vis Exp 122. https://doi.org/10.3791/55687
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Lee, R.F., Li, ML., Figetakis, M., Sumigray, K. (2024). A Coculture System for Modeling Intestinal Epithelial-Fibroblast Crosstalk. In: Methods in Molecular Biology. Springer, New York, NY. https://doi.org/10.1007/7651_2024_544
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DOI: https://doi.org/10.1007/7651_2024_544
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Publisher Name: Springer, New York, NY